The purpose of this project is to identify and characterize genes involved in generating a specialized cell during its differentiation. The cell chosen for study is the amoeboid sperm of the nematode C. elegans because every cellular stage that appears during its differentiation can be genetically dissected, differentiation will occur in vitro and be gentically dissected, differentiation will occur in vitro and sufficient material for biochemical analysis is obtainable. Four of the over 30 genes known to affect spermatogenesis in C. elegans have been selected for further genetic, cytological and molecular analysis. Each of these four genes arrests development at a different cytological stage and will allow analysis of the intracellular events at these stages of spermatogenesis. The specific aims are: (1) to isolate additional alleles of these four spermatogenesis defective (spe) mutants especially rearrangements and transposon insertions that will aid in their molecular analysis; (2) to build on the existing phenotypic data to identify how these 4 genes alter sperm morphogenesis; (3) to search for suppressors of these 4 genes to determine how they interact with other sperm proteins; (4) to clone these 4 spe genes taking advantage of the growing genomic physical map (already 90 percent) of overlapping cosmid clones and/or transposon insertion mutants whenever possible. Transgenic worms will be created to confirm that the sep gene has been cloned. The sequence of the cloned gene might suggest its function and will permit generation of an antibody, allowing additional cytological analysis of these 4 spe genes. This project has general significance because it is likely that the principles of cellular differentiation are similar in all organisms. Genetic methods can be used to define and alter parameters that control cell shape and the assymetric partitioning of cellular components during division in this system, phenomena that are clearly abnormal in cancerous cells but are very difficult to analyze. This research will also be useful for the study of spermatogenesis in mammals where both limited genetics and association of spermatocytes with the Sertoli cell makes it difficult to identify and study sperm specific genes.